Calcofluor

JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 1180-1185

0021-9193/85/091180-06$02.00/0 Copyright © 1985, American Society for Microbiology

Vol. 163, No. 3

Effect of Calcofluor White and Congo Red on Fungal Cell Wall Morphogenesis: In Vivo Activation of Chitin Polymerization
CESAR RONCERO AND ANGEL DURAN* Instituto de Microbiologia Bioquimica, Facultad de Biologia, Universidad deSalamancalConsejo Superior de Investigaciones Cientificas, Salamanca, Spain
Received 27 March 1985/Accepted 17 June 1985

Calcofluor White (a fluorochrome dye) affected the growth of Geotrichum lactis by causing lysis of cells at the hyphal tips. This effect was prevented by the presence of an osmotic stabilizer. The growth of Saccharomyces cerevisiae was also affected, and multicellular aggregates wereformed because of incomplete separation of mother and daughter cells; fluorescence microscopy indicated the presence of abnormally thick septa. The formation of rudimentary wail material by G. lactis protoplasts was promoted by Calcofluor or Congo red (another dye). The rate of chitin synthesis in protoplasts and growing cells was enhanced by both dyes. In contrast, both dyes inhibited chitin andj0(1,3)-glucan synthases in isolated cell-free systems. Degradation rates for I0(1,3)-glucan or chitin were not affected significantly by the dyes. Wheat germ agglutinin also affected chitin synthesis in protoplasts.

The fluorescent brightener Calcofluor White M2R New is used commercially to whiten textiles and paper and has been used as a stain for cell wall materials in fungi, algae, and higherplants (9, 15, 17, 22). This fluorochrome and another dye, Congo red, interact with various polysaccharides (31) but exhibit a particularly high affinity for chitin and cellulose (6, 14, 16). Through hydrogen bonding (with free hydroxyl groups [19] and dipolar interactions [26]) Calcofluor binds to nascent microfibers of cellulose in Acetobacter xylinum (14) and algae within the genus Oocystis(27). As a consequence of dye interaction the glucan chains are prevented from cocrystallizing to form microfibrils, and their assembly is thus seriously disrupted (3). Similar results have been described for chitin microfibril formation in the alga Poteriochromonas sp. with both Calcofluor and Congo red (16). Recent work (11, 29) has indicated that the assembly of chitin microfibrils in yeasts isalso altered by Calcofluor or Congo red. Furthermore, the in vitro inhibition of Neurospora crassa chitin synthase by Calcofluor has been reported (27). In this study we investigated the general effects of Calcofluor and Congo red on fungal wall morphogenesis. The effects of the dyes on the biosyntheses of two structural polysaccharides, ,(1,3)-glucan and chitin, during cell growth and protoplastwall generation were particularly explored. The results indicate that Calcofluor inhibits Geotrichum lactis growth but activates the synthesis of wall material by protoplasts. In the case of Saccharomyces cerevisiae, Calcofluor induces abnormal septa which apparently fail to develop abscission zones between mother and daughter cells. A similar effect was produced in S. cerevisiae by Congo red (30).The most notable finding in every case studied was that the rate of chitin polymerization was specifically enhanced in vivo by either Calcofluor or Congo red. This result confirms previous assumptions about the activation of chitin synthesis by these dyes in S. cerevisiae (12, 30) and is in concert with observed increases (in the presence of Calcofluor) of cellulose polymerization in A. xylinum(3). Preliminary results indicated that the lectin
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wheat germ agglutinin (WGA) also activates chitin synthesis in G. lactis protoplasts. MATERIALS AND METHODS Organisms and growth conditions. The filamentous fungus G. lactis ATCC 48590 was grown in 1% glucose-1% yeast extract (YED; Difco Laboratories, Detroit, Mich.); growth was quantified as described previously (23). The growth of S....